1. Field of the Invention
The present invention relates to hepatitis A virus and to a cellular receptor for hepatitis A virus. Specifically, the invention relates to nucleic acids encoding the receptor polypeptide, to the receptor polypeptide, and to homologs of the cloned hepatitis A virus receptor. The invention also relates to methods for detecting, preventing and treating hepatitis A virus infection, methods of purifying and removing hepatitis A virus from samples, methods of determining the anti-hepatitis A virus activity of compounds, transgenic animals expressing a foreign gene encoding the hepatitis A virus receptor, and methods of vaccine testing utilizing the cloned hepatitis A virus receptor expressed in a transgenic animal.
2. Background Art
Hepatitis A virus (HAV), a member of the Picornavirus genera, is a nonenveloped virus ranging from 27 to 32 nm in diameter. The virion of HAV is composed of a genome of a linear, single-stranded RNA of 7,480 nucleotides and a capsid containing multiple copies of three or four proteins. The genome of HAV can be divided into three parts: (1) a 5' noncoding region; (2) a single open-reading frame that appears to encode all of the viral proteins, with regions designated as P1 for capsid proteins and P2 and P3 for nonstructural proteins; and (3) a short 3' noncoding region. Hepatitis A virus is relatively acid stable, heat resistant, and generally resistant to harsh environmental conditions (Sobsey, M. D., et al., (1988)).
Natural infection with HAV usually follows ingestion of material contaminated with feces containing HAV. The course of viral hepatitis is variable: in addition to subclinical cases, patients may develop icteric or anicteric (jaundice associated or non-jaundice associated, respectively) hepatitis. Symptoms for HAV infection may range from mild and transient to severe and prolonged for the later two subgroups. Patients with subclinical hepatitis have neither symptoms nor jaundice; their disease is recognized by detecting biochemical or serological alterations in their blood. The frequency of clinical disease increases with age. Anicteric hepatitis A infection occurs in over 90% of infected children under the age of 5, whereas only 25 to 50% of infected adults respond to HAV infection without symptoms of jaundice. In contrast, icteric disease (jaundice) is very common in adults who acquire HAV.
Hepatitis A virus occasionally causes acute liver disease in infected individuals. In these occasional acute viral hepatitis, extensive necrosis of the liver may occur which leads to severe impairment of hepatic synthesis processes, excretory functions, and detoxifying mechanisms. Symptoms of this infection pattern are characterized by the sudden onset of high fever, marked abdominal pain, vomiting, and jaundice, followed by the onset of hepatic encephalopathy associated with deep coma and seizures. Ascites and decerebrate rigidity may lead to death in 70 to 90% of these patients.
Although the natural infection route for HAV is fecal-oral, it is still unclear whether the virus is transported directly to the liver or whether it undergoes primary replication at sites earlier in the infection route. Identification of primary infection sites is important in the development of prevention and effective therapeutic strategies and development of successful vaccines.
Currently there is no small animal model for studying hepatitis A virus infection, tropism, or disease progression in a laboratory setting. Large animal models are available, the chimpanzee for example, but such models are inherently limiting and constitute a significant barrier to effectively studying hepatitis A virus disease and the development of therapies, drugs, or vaccines to treat or prevent hepatitis A virus disease. A need therefore exists to develop a small animal model to provide a valuable research tool for economically and effectively studying hepatitis A virus infection and disease.
The cellular receptor is the major determinant of cell and tissue tropism for HAV. Identification of the cellular receptor for HAV is therefore important for determining the sites of primary infection and tissue tropism. Despite the known pathogenicity of HAV and the urgency of the development of a successful vaccine against HAV infection, a cellular receptor for HAV has not been identified in the art. Previous studies have only partially characterized various biochemical aspects of hepatitis A virus binding proteins, such as calcium dependency (Stapleton, J. T., et al., (1991)), the binding co-operativity of HAV to a HAV binding protein (Collier, A. J. and Wolstenholme, A. J. (1994)), and cell surface susceptibility of HAV attachment (Seganti, L., et al., (1987)). Anderson, et al., (abstract NO. P5-1 to the Third International Symposium on Positive Strand RNA Viruses, held in Clearwater, Fla. from Sep. 19 to Sep. 24, 1992) describes a protein stated to bind hepatitis A virus. Anderson, et al. states that HAV binds to a cell surface protein of molecular weight between 100,000 and 200,000 daltons. However, this protein is not a hepatitis A virus receptor. Thus, despite the need for a purified hepatitis A virus receptor, there has been no success.
Therefore a need exists to identify the cellular receptor for hepatitis A virus and to provide methods for diagnosing, treating, and preventing hepatitis A virus infection. Likewise, a need exists for methods for purifying and/or removing hepatitis A virus from samples, and for determining the anti-HAV binding activity of compounds. There also exists a need for the nucleotide sequence encoding the hepatitis A virus receptor and for transgenic animals expressing the exogenous hepatitis A virus receptor gene in order to effectively study the route of HAV infection and to develop successful vaccines to prevent against, or treatments for, HAV infection.